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Subcellular localization of the hum isoprenoid biosynthesis pathway

Hogenboom, S.

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Citation for published version (APA):Hogenboom, S. (2004). Subcellular localization of the hum isoprenoid biosynthesis pathway.

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Download date: 29 Mar 2020

HUMA NN MEVALONAT E PYROPHOSPHATE DECARBOXYLASE IS EXCLUSIVELYY LOCALIZED IN THE CYTOSOL

Sietskee Hogenboom, John J. M. Tuyp, Marc Espeel, Janet Koster, Ronald J. A. Wanderss and Hans R. Waterham (2003) Submitted for publication

ChapterChapter 6

HUMANN MEVALONATE PYROPHOSPHATE DECARBOXYLASE IS EXCLUSIVELYY LOCALIZED IN THE CYTOSOL

Sietskee Hogenboom1, John J. M. Tuyp1, Marc Espeel2, Janet Koster1, Ronald J. A. Wanders11 and Hans R. Waterham1

'Laboratory'Laboratory Genetic Metabolic Diseases, Departments of Clinical chemistry and Pediatrics/EmmaPediatrics/Emma Children's Hospital, Academic Medical Center, Amsterdam, The Netherlands Netherlands departmentdepartment of Anatomy, Embryology, Histology & Medical Physics, University of Gent, Belgium Belgium

SUMMARY Y

Thee importance of the cholesterol/isoprenoid biosynthetic pathway for humann development and health is emphasized by the multiple morphogenies, developmentall and neurological abnormalities associated with inborn errors inn this pathway. The subcellular localization of the pathway is a controversial subject.. In the past decade several reports have suggested that peroxisomes playy a critical role in the pathway since several of the enzymes involved have beenn reported to be localized predominantly in these organelles. However, otherr reports do not support this peroxisomal localization. In this study, the subcellularr localization of one of the enzymes, human mevalonate pyrophosphatee decarboxylase, was studied by conventional subcellular fractionationn and digitonin permeabilisation studies, immunofluorescence microscopyy and immunoelectron microscopy. An exclusive cytosolic localizationn was found for both endogenous human mevalonate pyrophosphatee decarboxylase (in human fibroblasts, liver and HEK293 cells) andd overexpressed mevalonate pyrophosphate decarboxylase (in human fibroblasts,fibroblasts, HEK293, CVi cells). We did not obtain any indication for a peroxisomall localization. Our results do not support a central role of peroxisomess in the cholesterol/isoprenoid biosynthetic pathway.

INTRODUCTION N

Mevalonatee pyrophosphate decarboxylase (MPD; E.C. 4.1.1.33) is one of the early enzymes off the cholesterol/isoprenoid biosynthetic pathway which provides the cell with the isoprenee unit isopentenylpyrophosphate (IPP). IPP is the building block of a wide variety off sterols and non-sterol isoprenoids that are involved in diverse cellular processes, includingg cell growth and differentiation, glycosylation, signal transduction, and electron transportt (1).

Thee prevailing view with respect to the subcellular distribution of the enzymes involved inn the cholesterol/isoprenoid biosynthesis is that peroxisomes, subcellular organelles

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implicatedd in a variety of metabolic processes, play a central role in the early, pre-squalene partt of the pathway (2). This view is based on various reports indicating that several enzymess involved in the conversion of acetyl-CoA into IPP are partly or even predominantlyy located in peroxisomes (3, 4, 5, 6, 7).

MPDD catalyzes the sixth reaction of the isoprenoid/cholesterol biosynthetic pathway whichh is the decarboxylation and hydration of mevalonate pyrophosphate to produce IPP. Thee postulation that MPD would be located in peroxisomes is based on the following observations.. Firstly, in some livers of patients suffering from Zellweger syndrome, which doo not contain peroxisomes, a 60% decrease in MPD activity was found (8). Since peroxisomee deficiency often leads to degradation and/or inactivation of peroxisomal enzymess as a result of the mislocalization to the cytosol, this decreased activity has been explainedd to be indicative of a peroxisomal localization of MPD. Secondly, latency of MPD activityy was found in monkey kidney (CVi) cells after selective permeabilisation of cellular membraness with digitonin (6). This latency was similar to the latency of the peroxisomal catalase,, suggesting that both enzymes are located in the same subcellular compartment. Thirdly,, immunofluorescence microscopy performed with CHO cells and peroxisomal targetingg sequence (PTS) 1 protein import-deficient human fibroblasts, transiently transfectedd with an amino-terminal fragment (47 amino acids) of human MPD tagged with ann HA epitope revealed a punctate pattern similar to the distribution of the peroxisomal catalasee suggesting that the amino terminus harbours a PTS2-like PTS (9). Indeed, transientt transfection of this construct in PTS2 protein import-deficient fibroblasts resultedd in a cytosolic localization, suggesting that the construct is imported into peroxisomess via the PTS2 receptor protein PEX7 (9). Finally, transient transfection of the samee construct lacking the SVX5QL sequence resulted in a cytosolic localization indicating thatt this sequence might be a novel PTS2-like signal (9).

Otherr data, however, do not support a peroxisomal localization of MPD. Firstly, we measuredd normal MPD activity and MPD protein levels in fibroblasts and liver homogenatess of patients with a peroxisome biogenesis defect and in liver homogenates of PEX5PEX5 knock-out mice (10, 11). Moreover, also in PTS2 protein-import deficient cells from patientss affected with rhizomelic chondrodysplasia punctata no deficient activity was observedd (unpublished data). Secondly, selective permeabilization with digitonin of rat hepatocytes,, normal rat kidney cells or mouse melanoma (B16F10) cells resulted in a completee release of MPD protein similar to the release of the cytosolic lactate dehydrogenase,, suggesting that both enzymes are in the same subcellular compartment. Underr these conditions peroxisomal catalase activity was completely retained in the cells (12,, 13). Thirdly, after subcellular fractionation of B16F10 cells, the MPD protein was recoveredd in fractions which contained only cytosolic (and no peroxisomal) proteins indicatingg that MPD is a cytosolic protein (13). Furthermore, immunofluorescence microscopyy in rat hepatocytes using purified antibodies directed against rat MPD showed a cytosolicc localization of the MPD protein (12). Finally, in conventional subcellular fractionationn studies performed with HepG2 cells, we never have been able to demonstrate aa peroxisomal localization of MPD activity (unpublished data).

Inn summary, from the combined data one may conclude that MPD is a cytosolic protein inn rat and mouse cells while in human cells the subcellular localization is not clear. So far, thee subcellular distribution of human MPD has been studied mostly in overexpression

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systemss using constructs containing only the ami no-terminal part of MPD. However, it remainss to be determined whether the authentic human protein is a peroxisomal or cytosolicc protein or both. This prompted us to initiate a thorough study to conclusively determinee the subcellular localization of human MPD both under normal conditions and whenn overexpressed in cell lines. Using a variety of biochemical and microscopical techniques,, we found a cytosolic localization of both endogenously expressed and overexpressedd MPD and no indication of a peroxisomal localization.

MATERIALSS AND METHODS

CellCell lines and culture conditions Primaryy skin fibroblasts were obtained from a healthy control subject, from a patient who sufferedd from Zellweger syndrome and who was a homozygote for an insertion mutation in thee PEX19 gene (14), and from a patient affected with homozygous familial hypercholesterolemiaa (FHC) (GM00701, Coriell cell repositories). The fibroblasts were culturedd in HAM F-10 containing 10% FCS and 1% penicillin/streptomycin in a temperaturee and humidity controlled incubator (95% air, 5% C02 as the gas phase) at 37°C.. Prior to experiments the cells were grown until 70-80% confluency after which the mediumm was substituted with HAM containing 10% lipoprotein (cholesterol)-depleted fetal calff serum. Experiments were performed after 72 hours of culturing in lipoprotein (cholesterol)-depletedd medium.

Forr MPD expression studies, the HEK293 Flp-In and CVi Flp-In cell lines (Invitrogen) weree used and cultured in DMEM containing 10% FCS, 1% penicillin/streptomycin and 1000 ng/ml hygromycin in a temperature and humidity controlled incubator (95% air, 5% C022 as the gas phase) at 37°C. Prior to experiments the cells were grown until 70-80% confluencyy after which the medium was substituted for DMEM containing 10% lipoprotein (cholesterol)-depletedd fetal calf serum. Experiments were performed after 24 hours of culturingg in lipoprotein (cholesterol)-depleted medium.

GenerationGeneration of cell lines stably overexpressing human MPD Thee open reading frame (ORF) of human MPD cDNA was amplified by PCR from cDNA preparedd from human skin fibroblasts and ligated as a BamHl-XhoI fragment under controll of the CMV promoter in the pcDNAs/FRT vector (Invitrogen). The entire insert wass sequenced to assure the absence of taq polymerase-introduced errors.

HEK2933 Flp-In cells or CVi Flp-In cells were cultured in Dulbecco's modified Eagle's mediumm (DMEM), containing 10% fetal calf serum (FCS), 1% penicillin/streptomycin. Stablee MPD-expressing cell lines were generated by co-transfection of the POG44 and PCDNA5/FRT-MPDD (CVi and HEK293) using lipofectamine plus reagent in growth mediumm without zeocin according to the manufacturer's recommendations (Invitrogen). Forty-eightt hours after transfection, hygromycin B was added to the medium to a final concentrationn of 100 ug/ml, and the media were changed every 3-4 days until hygromycin-resistantt colonies were evident. Control hygromycin-resistant cell lines were generated by co-transfectionn of the POG44 with the empty pcDNAs/FRT vector. For expression studies thee HEK293 Flp-In cell lines, stably expressing human MPD (HEK-MPD), CVi Flp-In cell

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liness stably expressing human MPD (CVi-MPD) and the control cell lines transfected with emptyy pcDNAs/FRT (HEK- or CVi-) were cultured in DMEM containing 10% FCS, 1% penicillin/streptomycinn and 100 ug/ml hygromycin. The activity in cells overexpressing humann MPD was 5 times higher compared to the control cell lines.

SubcellularSubcellular fractionation Forr subcellular fractionation studies, cells were cultured in 162-cm2 Falcon flasks, harvested,, washed three times with PBS and two times with fractionation buffer (0.25 M sucrose,, 1 mM EDTA, 10 mM HEPES, 1 mM phenylmethylsulfonyl fluoride, pH=7-4). Next, thee cells were homogenized using a ball bearing cell cracker (EMBL, Germany), after which thee post nuclear supernatant (PNS, 10', 500 x g) was layered on top of a continuous nycodenzz gradient (15-35%), with a cushion of 1 ml 50% nycodenz in 0.25 mM sucrose, 5 mMM MOPS, 1 mM EDTA, and 2 mM KC1 (pH=7-3). Gradients were centrifuged for 2.5 hourss in a vertical rotor (MSE 8x35) at 19,000 rpm at 4°C. After centrifugation, 16-19 fractionss were collected from the bottom of the gradient.

CellCell permeabilization with digitonin Celll permeabilization experiments were performed essentially as described by Biardi et al. (6)) with a few modifications. HEK293 and CV-i cells were seeded in 60 mm plates at a densityy of 3.0 x IOS cells/plate and fibroblast cells at a density of 2.0 x ios cells/plate. After culturingg for one or three days in DMEM or HAM containing 10% lipoprotein (cholesterol)-depletedd fetal calf serum, cells were washed twice with ice-cold KH buffer (50 mMM HEPES, 110 mM KOAc, pH=7.2). The plates were then transferred on ice and incubatedd in KHM buffer (20 mM HEPES, 110 mM KOAc, 2 mM MgOAc, pH=7.2) containingg various concentrations of digitonin (o, 20, 50, 150, 500 or 1000 ug/ml) or, as a control,, 0.1% (w/v) triton X-100. After 5 minutes, the buffer was collected as supernatant fractionss and kept on ice. Subsequently, cells were incubated in KH buffer containing 1000 ug/mll digitonin. After 30 minutes, the buffer was collected as pellet fractions and kept on ice.. Enzyme measurements were done immediately in both fractions.

EnzymeEnzyme assays MPDD activity was measured radiochemical̂ as described previously (10). Phospho gluco isomerasee (PGI) (15) and catalase (CAT) (8) activities were measured spectrophotometricallyy as described.

ImmunoblotImmunoblot analysis Proteinss were separated by SDS-PAGE and transferred onto nitrocellulose by semidry blottingg (16). The highly specific affinity-purified antibody directed against human MPD (10)) was used at a 1:250 dilution. Antigen-antibody complexes were visualized with goat anti-rabbitt IgG-alkaline phosphatase conjugate and CDP-star. As a control for transfer of protein,, each blot was reversibly stained with Ponceau S prior to the incubation with antibodies. .

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66 7 99 101112131415161718 fractionn number

77 8 9 101112131415161718 66 7

99 101112131415 fractionn number 99 101112131415

161718 8

161718 8

00 20 50 150 500 1000 triton concentrationn digitonin (ug/ml)

r - m m

140 0 -20 0

11 o 00 20 50 150 500 1000 triton concentrationn digitonin (ug/ml)

relativee concentration digitonin (ug/ml) supernatantt pellet

relativee concentration digitonin (ug/ml) supernatantt " pellet

Figuree l . Subcellular fractions of human fibroblasts derived from a control subject (A) or a ZS

patientt (B) were obtained by nycodenz equilibrium density gradient centrifugation as described

inn materials and methods. Fractions were analyzed for the cytosolic marker PGI (black bars) and

thee peroxisomal marker CAT (gray bars). Relative activities were expressed as a percentage of

totall gradient activity present in each fraction. The pattern of distribution of MPD protein as

determinedd by immunoblot analysis with an affinity purified antibody raised against human

MPDD is similar to the pattern of PGI activity.

Humann fibroblasts derived from a control subject (C) or a ZS patient (D) were incubated with

increasingg concentrations of digitonin as described in materials and methods. Supernatant

(closedd symbols) and pellet (open symbols) fractions were analyzed for the activities of the

cytosolicc marker PGI (square) and the peroxisomal marker CAT (triangle). Relative activities

weree expressed as a percentage of total activity (supernatant + pellet) present in each fraction.

Thee pattern of latency of MPD protein as determined by immunoblot analysis with an affinity

purifiedd antibody raised against human MPD is similar to the pattern of PGI activity.

Immunofluorescence Immunofluorescence Cellss were seeded on cover slides in 6 wells-plates and cultured as indicated in Cell lines andand culture conditions. Immunofluorescence was performed as described (17). Cells were doublee labeled with antibodies directed against human MPD (10) and the peroxisomal markerr catalase (18) or the cytosolic marker metallo matrix protein 7 (MMP7) (MMP-7 Ab-11 (Clone 1D2), Labvision). MPD antibodies were visualized using biotinylated Donkey-anti-Rabbitt Ig (Amersham) and streptavidin-labeled fluorescein isothiocyanate (Strep-Fitc). Catalasee and MMP7 were visualized using Goat-anti-Mouse-labeled Alexa568 (Molecular Probes).. Pictures were taken using a confocal laser scanning microscope (Leica).

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LiverLiver immunoelectron microscopy Humann liver biopsies were fixed in 4% formaldehyde in 0.1 M sodium cacodylate buffer (pH=7-3)) containing 1% calcium chloride and processed for Unicryl embedding as describedd (19). Ultra thin sections of Unicryl embedded samples were immunostained with polyclonall antibodies against MPD (10) or the peroxisomal alanine/glyoxylate aminotransferasee (AGT) (20) as previously described (19). Negative controls were incubatedd with normal rabbit serum.

RESULTS S

SubcellularSubcellular fractionation of MPD in humanfibroblasts Too determine whether human MPD is localized in the cytosol or the peroxisomes or both, wee first performed subcellular fractionation studies with human skin fibroblasts. As a controll we included fibroblasts from a patient who suffered from Zellweger syndrome. Previouss studies have demonstrated that in this particular cell line no peroxisomal remnantss are present due to a homozygous insertion in the PEXig gene (14). After growth off the cells in lipoprotein-depleted medium to assure optimal induction of the isoprenoid biosyntheticc pathway, we prepared a post-nuclear supernatant (PNS) which was further fractionatedd by nycodenz equilibrium density gradient centrifugation. In the normal fibroblasts,fibroblasts, this resulted in a clear separation of peroxisomes and cytosol as reflected by thee distribution of the peroxisomal marker enzyme catalase and the cytosolic marker enzymee PGI (Fig. lA) . In the ZS fibroblasts, both marker enzymes colocalize as expected fromm the absence of peroxisomes, which leads to the cytosolic localization of peroxisomal enzymess (Fig. lB). Immunoblot analysis of the fractions from the same density gradients usingg affinity-purified antiserum against human MPD revealed a similar distribution patternn for MPD protein as for PGI activity (Fig. lA, B).

DigitoninDigitonin permeabilization studies in humanfibroblasts Ass an alternative approach to study the subcellular localization of MPD in human fibroblastss we exposed the cells to increasing concentrations of digitonin. Digitonin permeabilizess cellular membranes by complexing with cholesterol. Since the membranes of mostt cellular organelles contain lower levels of cholesterol than the plasma membrane, cellss wil l lose their cytosolic components at lower concentrations of digitonin than the organellarr contents. Indeed, when we measured the enzyme activities of CAT and PGI in supernatantt and pellet fractions of normal fibroblasts we found a clearly increased latency forr CAT compared to PGI (Fig. lC). This indicated that the plasma membrane was disruptedd first, resulting in the release of cytosolic PGI. The peroxisomal membranes, however,, are only permeabilized at higher concentrations of digitonin resulting in the releasee of the peroxisomal matrix content, including CAT, into the supernatant fraction. As expected,, in the ZS fibroblasts lacking peroxisomes, no difference in latency between PGI andd CAT was observed (Fig. lD). When we determined MPD protein by immunoblot analysiss in all pellet and supernatant fractions, we found that the release of MPD from the normall fibroblasts into the supernatant fractions occurs at the same concentration of digitoninn as cytosolic PGI (Fig. lC). In the ZS fibroblasts, PGI, CAT and MPD were released

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fromm the cells at the same digitonin concentration (Fig. lD). Thus, also in digitonin permeabilizationn studies human MPD behaves similar as cytosolic PGI and clearly differentt than peroxisomal CAT.

ImmunofluorescenceImmunofluorescence studies infibroblasts Too study the subcellular localization of human MPD further, we performed immunofluorescencee microscopy making use of the highly specific affinity-purified polyclonall antibodies directed against human MPD. To this end, we performed double labelingg of fibroblasts cultured in lipoprotein-depleted medium using the anti-MPD antiserumm and a monoclonal antibody directed against human peroxisomal CAT or a monoclonall antibody directed against human MMP7, a cytosolic marker (Fig. 2). When we comparedd the immunolabeling of MPD in the normal fibroblasts and ZS fibroblasts, we observedd a similar cytosolic distribution pattern of the fluorescent signal in both cell lines, indicatingg that the presence or absence of peroxisomes does not affect the localization of MPD.. Moreover, there was no co-localization of MPD and CAT in the normal fibroblasts whilee in the ZS fibroblasts the distribution pattern of CAT is superimposable to that of MPDD indicating a co-localization of CAT and MPD in the cytosol. Also when we compared thee fluorescent signals obtained with anti-MPD and anti-MMP7 we found a clear colocalizationn in the cytosol both in normal fibroblasts and ZS fibroblasts.

Figuree 2. Human fibroblasts derived from a control subject (A-D) or a ZS patient (E-H) were labeledd with antibodies as described in materials and methods. Cells were double labeled using antibodiess directed against MPD (A, E) and the peroxisomal marker CAT (B, F) or with antibodiess directed against MPD (C, G) and the cytosolic marker MMP7 (D, H). MPD shows the samee pattern as the cytosolic MMP7 in both cell lines. MPD shows co-localization with CAT in thee ZS fibroblasts in which CAT is localized in the cytosol but no co-localization is observed betweenn MPD and the peroxisomal CAT in control fibroblasts.

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SubcellularSubcellular localization of human MPD in overexpressing cell lines Thee results of the various localization studies in human fibroblasts all indicate that endogenouss MPD is at least predominantly, if not exclusively, located in the cytosol and nott in peroxisomes. These result are in apparent contrast to the reported peroxisomal localizationn of an overexpressed amino-terminal fragment of 47 amino acids of the human MPDD fused with an HA epitope upon transient transfection into human fibroblasts and CHOO (9). To determine whether this discrepancy in localization might be due to a differencee in expression levels, we decided to also investigate the subcellular localization of overexpressedd MPD in various cell types. These include CVi and HEK293 cells stably transfectedd with human MPD cDNA under control of the CMV promotor and human

Figuree 3. Subcellular fractions of human fibroblasts derived from an FHC patient (A), HEK2933 (B) cells or CVi cells (C) overexpressing full length human MPD were obtained by nycodenzz equilibrium density gradient centrifugation as described in materials and methods. Fractionss were analyzed for the cytosolic marker PGI (black bars) and the peroxisomal marker CATT (gray bars). Relative activities were expressed as a percentage of total gradient activity presentt in each fraction. The pattern of distribution of MPD protein as determined by immunoblott analysis with an affinity purified antibody raised against human MK is similar to thee pattern of PGI activity.

Humann fibroblasts derived from an FHC patient (D), HEK293 cells (E) or CVi cells (F) overexpressingg full length human MPD were incubated with increasing concentrations of digitoninn as described in materials and methods. Supernatant (closed symbols) and pellet (open symbols)) fractions were analyzed for the activities of the cytosolic marker PGI (square) and the peroxisomall marker CAT (triangle). Relative activities were expressed as a percentage of total activityy (supernatant + pellet) present in each fraction. The pattern of latency of MPD protein as determinedd by immunoblot analysis with an affinity purified antibody raised against human MPDD is similar to the pattern of PGI activity.

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fibroblastsfibroblasts from a patient homozygous for familial hypercholesterolemia, which exhibit fivefive times higher MPD activity when compared to control fibroblasts (Hogenboom et al., unpublishedd results).

Afterr fractionation of the various PNS fractions of these cell lines by nycodenz equilibriumm density gradient centrifugation followed by the measurement of PGI and CAT activitiess and MPD protein content in all fractions we found again a distribution pattern of MPDD similar to cytosolic PGI and clearly distinct of peroxisomal CAT in all cell lines (Fig. 3A-C).. This was the case for endogenously overexpressed human MPD (FHC (Fig. 3A)), constitutivelyy overexpressed human MPD (HEK-MK; Fig. 3B, CVi-MK ; Fig. 3C), endogenouslyy expressed human MPD (HEK- cells; not shown) and monkey MPD (CVi-cells;; not shown). Also after selective permeabilization of the cellular membranes using increasingg concentrations of digitonin, we found that both endogenously and constitutively overexpressedd human MPD behaves similar as cytosolic PGI (Fig. 3D-F). Moreover, immunofluorescentt labeling of the endogenously and constitutively overexpressed MPD showss a cytosolic localization superimposable to that of cytosolic MMP7 protein, and clearlyy different from the localization of CAT in thesee cell lines (Fig. 4).

Figuree 4. Human fibroblasts derived from an FHC patient (A-D), HEK293 cells (E-H) or CVi cellss (I-L) overexpressing full length human MPD were labeled with antibodies as described in materialss and methods. Cells were double labeled using antibodies directed against MPD (A, E, I)) and the peroxisomal marker CAT (B, F, J) or with antibodies directed against MPD (C, G, K) andd the cytosolic marker MMP7 (D, H, L). The diffuse distribution pattern of MPD differs from thee punctated pattern of CAT but MPD shows the same pattern as the cytosolic MMP7 in all cell lines. .

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MevalonateMevalonate pyrophosphate decarboxylase is a cytosolic enzyme

ImmunoelectronImmunoelectron microscopy studies in human liver Whilee our combined data show that at least in humans, MPD is predominantly a

cytosolicc protein, they cannot exclude the possibility that a minor amount of MPD is localizedd in peroxisomes. Therefore, we also performed immunogold labelling on ultrathin sectionss of human liver, the organ displaying the highest expression of the enzymes of the presqualenee segment of the isoprenoid biosynthesis pathway. Inn the immunogold labelling experiments using the highly specific affinity-purified polyclonall antibodies directed against human MPD we found only occasional labelling in thee cytoplasm of the liver parenchyal cells. Although we carefully checked a large number off peroxisomes we were unable to detect any labelling of MPD in these peroxisomes (Fig. 5A).. Moreover, even after incubation with higher concentrations of antibodies, as a result off which aspecific labelling strongly increased, no peroxisomal labelling could be observed. Ass a control we performed immunogold labelling experiments on sections of the same sampless with antibodies against peroxisomal AGT. This revealed a distinct label which was foundd in the peroxisomal matrix (Fig. 5B), while no label was observed in negative controls. .

Figuree 5. Electron microscopy of human control liver. (A) Ultrathin Unicryl sections of human liverr were immunostained with the affinity purified antibodies against MPD. The peroxisomes (P)) remain unlabelled. (B) Ultrathin Unicryl sections immunostained with antibodies against againstt AGT reveals a clear localisation in the peroxisomal matrix. Scale bar = 500 nm; M, mitochondria. .

DISCUSSION N

Thee cholesterol/isoprenoid biosynthetic pathway has been the subject of extensive studies inn the last decades. The importance of the pathway for human development and health is underlinedd by the identification of patients with inborn errors in this pathway, which are oftenn characterized by multiple morphogenic, developmental and neurological

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abnormalities.. However, the subcellular localization of the enzymes involved in the pathwayy is still a very controversial subject. Various studies have suggested that, in additionn to enzymes involved in a variety of other metabolic pathways, peroxisomes predominantlyy harbor most of the enzymes involved in the presqualene part of the cholesterol/isoprenoidd biosynthetic pathway including 3-hydroxy-3-methylglutaryl CoA reductasee (3), mevalonate kinase (4), phosphomevalonate kinase (5), MPD (6), isopentenyl pyrophosphatee isomerase (7) and farnesylpyrophosphate synthase (8, 21).

Thee peroxisomal localization of the authentic MPD, however, has not been demonstratedd in humans, although a reporter construct consisting of an amino terminal fragmentt of MPD fused to an HA tag was reported to end up in peroxisomes. In contrast, full-lengthh MPD has been shown to be cytosolic in rat and mice (12, 13). To conclusively determinee the subcellular localization of human MPD and the relevance of peroxisomes in cholesterol/isoprenoidd biosynthesis, we used a variety of techniques to study the localizationn of the enzyme, including conventional subcellular fractionation and cell permeabilizationn techniques, immunofluorescence and electron microscopy techniques. Ourr results unequivocally demonstrate that both endogenous (in human fibroblasts, humann liver and HEK293 cells) and overexpressed full-length human MPD (in human fibroblasts,fibroblasts, HEK293 and CVi cells) have an exclusive cytosolic but no peroxisomal localization. .

Thiss conclusion is in line with our recent studies in which normal MPD activities and MPDD protein were found in patients suffering from peroxisome biogenesis disorders (10, 11)) and with the cytosolic localization of MPD in rat and mouse (12, 13). The fact that humann MPD is a cytosolic enzyme raises questions about the postulated central role of peroxisomess in cholesterol/isoprenoid biosynthesis. In fact, using a similar approach as usedd for our localization studies of MPD, we also could demonstrate a cytosolic localization off human mevalonate kinase and human phosphomevalonate kinase, two enzymes that hadd been postulated to be predominantly peroxisomal (manuscripts in preparation).

Thesee combined results imply that peroxisomes may not play a central role in cholesterol/isoprenoidd biosynthesis.

ACKNOWLEDGEMENTS S

Thee authors thank Dr. J. van Marie and H.A. van Veen for technical assistance with confocall laser scanning microscopy, D. Jacobus and G. Van Limbergen for assistance with immunolabellingg procedures and E. Heijkoop for assistance with the generation of cell liness stably overexpressing human MPD. This research was supported financially by a grantt of The Netherlands Organization for Scientific Research, division of Medical Sciences (NWO-MW).. Dr. H.R. Waterham is supported by a fellowship of the Royal Netherlands Academyy of Arts and Sciences.

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Krisans,, S. K. (1994) Mevalonate kinase is predominantly localized in peroxisomes and is defective in patientss with peroxisome deficiency disorders. J. Biol. Chem. 269: 1197-1205.

5.. Olivier, L. M., Chambliss, K. L., Gibson, K. M., and Krisans, S. K. (1999) Characterization of phosphomevalonatee kinase: chromosomal localization, regulation, and subcellular targeting. J. Lipid Res.Res. 40: 672-679.

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7.. Paton, V. G., Shackelford, J. E., and Krisans, S. K. (1997) Cloning and subcellular localization of hamster andd rat isopentenyl diphosphate dimethylallyl diphosphate isomerase. A PTSi motif targets the enzyme too peroxisomes. J. Biol. Chem. 272: 18945-18950.

8.. Krisans, S. K., Ericsson, J., Edwards, P. A., and Keller, G. A. (1994) Farnesyl-diphosphate synthase is localizedd in peroxisomes. J. Biol. Chem. 269: 14165-14169.

9.. Olivier, L. M., Kovacs, W., Masuda, K., Keller, G. A., and Krisans, S. K. (2000) Identification of peroxisomall targeting signals in cholesterol biosynthetic enzymes. AA-CoA thiolase, HMG-CoA synthase, MPPD,, and FPP synthase. J. Lipid Res. 41: 1921-1935.

10.. Hogenboom, S., Romeijn, G. J., Houten, S. M., Baes, M., Wanders, R. J., and Waterham, H. R. (2002) Absencee of functional peroxisomes does not lead to deficiency of enzymes involved in cholesterol biosynthesis.. J. Lipid Res. 43: 90-98.

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12.. Michihara, A., Sawamura, M., Yamori, Y., Akasaki, K., and Tsuji, H. (2001) Mevalonate pyrophosphate decarboxylasee is predominantly located in the cytosol of rat hepatocytes. Biol. Pharm. Bull. 24: 1235-1240. .

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